New Treatment for Mesothelioma Attacks Cancer Stem Cells


The term mesothelium refers to a membrane that forms the lining of several body cavities such as the pleural cavity that contains the lungs or the peritoneum that contains the gastrointestinal tract. Inhalation of asbestos fibers repeatedly injures the mesothelium, and the cycle of injury and regeneration selects for cells that grow faster and faster. Such conditions predispose people to cancer and cancer of the mesothelium or mesothelioma is a consequence of repeated exposure to asbestos fibers.

Mesothelium

In the United Kingdom (UK), asbestos was banned in 1985, but the number of asbestos-related deaths has climbed from 153 in 1968 to 2,321 in 2009 and epidemiologists have estimated that the number of asbestos-related deaths will continue to rise over the next 20 years, peaking in 2020.

Mesothelioma treatments do not provide a terribly good prognosis. The drug cisplatin alone or in combination with pemetrexed (brand name Alimta) or cisplatin in combination with raltitrexed, but raltitrexed is no longer commercially available for this type of treatment regime. Cisplatin has also been used in combination with gemcitabine or vinorelbine. If cisplatin cannot be used then carboplatin can be substituted. In all cases, survival rates are rather underwhelming.

The importance of this clinical issue to stem cell biology is that mesothelioma is a type of cancer driven by rogue stem cells that grow uncontrollably. To that end, Dean Fennell and his team from Leicester University, UK have conducted a clinical trial to test a new treatment for mesothelioma.

The Meso2 study, conducted by Synta Pharmaceuticals, examined the efficacy of a drug called ganetspib as a treatment for mesothelioma. The trial is being led by Fennell and his research team, and will enroll about 140 patients.

Ganetespib is a unique drug in that it targets a protein called HSP90 (heat shock protein 90). Heat shock proteins help proteins fold and help unfolded proteins refold. They are called heat shock proteins because their expression increases when temperatures are raised. Since cancer cells grow quickly and make large quantities of protein, hamstringing those proteins that fold other proteins can gum up the internal workings of the cell and cause them to die.

HSP90

Fennell said, “We think this is a new way to being able to target mesothelioma. Laboratory tests show ganetespib is extremely active in mesothelioma, and combined with chemotherapy, this treatment could shrink cancers down and improve symptoms for patients.”

There is also a second clinical trial called COMMAND (Control of Mesothelioma with MAintenance Defactinib) is being sponsored by a Verastem, a Cambridge, Massachusetts-based pharmaceutical company and it will test a new drug called defactinib.

Defactinib inhibits a protein called FAK (focal adhesion kinase), which is also crucial for cancer stem cell function and for the conversion of cancer stem cells into tumors.  FAK acts as an adapter between the cell adhesion molecules on the surface of the cell, and the internal skeleton proteins of the cell.  Therefore when the cell attaches to another cell or a substratum, FAK and the proteins associated with it transmits a message to the rest of the cell that the cell has attached to another cell.  For a great website about FAK, see here.

FAK

Defactinib inhibits FAK and prevents the cell from adapting to its environment and since FAK is involved with spread of the cell over its substratum, proliferation of the cell and migration of the cell.  Inhibition of FAK prevents the cell from properly responding to surface stimuli, and the cell stops growing.

The COMMAND trial will enroll some 350-400 people and Fennell’s lab is involved with starting this trial.

Cancer stem cells can cause cancer to return to return after chemotherapy because most chermotherapeutic strategies attack the progeny of cancer stem cells and not the cancer stem cells themselves.  Inhibiting the FAK protein takes away something cancer stem cells crucially need and Fennell hopes that treatments like defactinib or ganetespib will positively help mesothelioma patients.

Stem Cell Behavior in Three-Dimensional Matrices


Scientists from Case Western Reserve in Cleveland, Ohio have used hydrogels (jello-like materials) to make three-dimensional structures that direct stem cell behavior.

Physical and biochemical signals guide stem cell behavior and directs them to differentiate and make tissues like muscle, blood vessels, or bone. The exact recipes to produce each particular tissue remains unknown, but the Case Western Reserve team has provided a way to discover these recipes.

Ultimately, scientists would like to manipulate stem cells in order to repair or replace damaged tissues. They would also like to engineer new tissues and organs.

Eben Alsberg. associate professor of biomedical engineering and orthopedic surgery at Case Western Reserve, who was also the senior author on this research said, “If we can control the spatial preservation of signals, we have be able to have more control over cell behavior and enhance the rate and quality of tissue formation. Many tissues form during development and healing processes at least in part due to gradients of signals: gradients of growth factors, gradients of physical triggers.”

Alsberg and his colleagues have tested their system on mesenchymal stem cells, and in doing so have turned them into bone or cartilage cells. Regulating the presentation of certain signals in three-dimensional space may be a key to engineering complex tissues; such tissues as bone and cartilage. For example, if we want to convert cartilage-making cells into bone-making cells or visa-verse, several different signals are required to induce the stem cells to change into different cell types in order to form the tissues you need.

To test their ideas, Alsberg and coworkers two different growth factors directed the stem cells to differentiate into either bone or cartilage.  One of these growth factors, transforming growth factor-beta (TGF-beta) promotes cartilage formation while a different growth factor, bone morphogen protein-2 (BMP-2).  Alsberg and his crew placed mesenchymal stem cells into an alginate hydrogel with varying concentrations of these growth factors.  Alginate comes from seaweed and when you hit it with ultraviolet light, it crosslinks to form a jello-like material called a hydrogel.   To create gradients of these growth factors, Alsberg developed a very inventive method in which they loaded a syringes with these growth factors and hooked them to a computer controlled pump that released lots of BMP-2 and a little TGF-1beta and tapered the levels of BMP-2 and then gradually increased the levels of TGF-1beta (see panel A below).  

 Fabrication of microparticle-based gradient alginate hydrogels. (A) Photograph of gradient making system. (B) Flow rates of two syringes to pump a linear gradient for a 5 cm length × 2 mm diameter alginate hydrogel. After linear gradient pumping for 3 min, an additional 50 μL of alginate solution, which is the volume from the Y point to the beginning of quartz tube, was further pumped into a spiral mixer for 1 min. (C) Photomicrographs of microparticles in cross-sections of gradient alginate hydrogel segments. Segments 1-10 represent sequential segments of the gel. (D) Quantification of microparticles in each segment of gradient alginate hydrogels.
Fabrication of microparticle-based gradient alginate hydrogels. (A) Photograph of gradient making system. (B) Flow rates of two syringes to pump a linear gradient for a 5 cm length × 2 mm diameter alginate hydrogel. After linear gradient pumping for 3 min, an additional 50 μL of alginate solution, which is the volume from the Y point to the beginning of quartz tube, was further pumped into a spiral mixer for 1 min. (C) Photomicrographs of microparticles in cross-sections of gradient alginate hydrogel segments. Segments 1-10 represent sequential segments of the gel. (D) Quantification of microparticles in each segment of gradient alginate hydrogels.

The result has an alginate hydrogen with mesenchymal stem cell embedded in it that had a high concentration of BMP-2 at one end and a high concentration of TGF-1beta at the other end.  Alsberg also modified the hydrogel by attached RGD peptides to it so that the stem cells would bind the hydrogel.  The peptide RGD (arginine-glycine-aspartic acid) binds to the integrin receptors, which happen to be one of the main cell adhesion protein on the surfaces of these cells.  This modification increases the exposure of the mesenchymal stem cells to the growth factors.  After culturing mesenchymal stem cells in the hydrogel, they discovered that the majority of the cells were in the areas of the hydrogel that had the highest concentration of RDG peptides.  

In another other experiment Alsberg and others varied the crosslinks in the hydrogel.  They used hydrogels with few crosslinks that were more flexible and hydrogels that have quite a few crosslinks and were stiffer.  The stem cells clearly preferred the more flexible hydrogels.  Alsberg thinks that the more flexible hydrogels might show better diffusion of the growth factors and better waste removal.  

“This is exciting,” gushed Alsberg.  “We can look at this work as a proof of principle.  Using this approach, you can use any growth factor or any adhesion ligand that influences cell behavior and study the role of gradient presentation.  We can also examine multiple different parameters in one system to investigate the role of these gradients in combination on cell behavior.”  

This technology might also be a platform for testing different recipes that would direct stem cells to become fat, cartilage, bone, or other tissues.  Also, since this hydrogel is also biodegradable, stem cells grown in the hydrogel could be implanted into patients.  Since the cells would be in the process of forming the desired tissue, their implantation might restore function and promote healing.  Clearly Alsberg is on to something.  

New Drug Prevents Viral Infections in Stem Cell Transplant Patients


Because bone marrow transplant patients have had their bone marrows wiped out with radiation or rather severe drugs, their immune systems tend to be kaput until the transplanted bone marrow stem cells start making new immune cells to reconstitute the immune system. Consequently, bone marrow transplant patients can contract a whole host of truly diabolical diseases.

One disease that shows up with some frequency in bone marrow transplant patients is cytomegalovirus (CMV) infections. CMV can cause pneumonia, diarrhea, digestive tract ulcers, and other problems. Some antiviral drugs do exist (ganciclovir, or its prodrug valganciclovir, foscarnet, and cidofovir), but they can cause kidney dysfunction or bone marrow suppression. Neither of these are desirable side effects. Clearly new drugs are needed (see Ahmed, A. Infect Disord Drug Targets. 2011 Oct;11(5):475-503).

A new clinical trial by researchers at Dana-Farber Cancer Institute and Brigham and Women’s Hospital has tested a drug called CMX001. When bone marrow transplant patients took it shortly after transplant, they were much less likely to contract CMV infections that those who did not take the drug.

The study’s lead author, Francisco Marty from Dana-Farber and Brigham and Women’s said: “With current agents, between 3 and 5 percent of allogeneic transplant patients develop CMV disease within six months of transplantation, and a small number of them die of it. There is clearly a need for better treatments with fewer adverse effects. This clinical trial examined whether the disease can be prevented, rather than waiting for blood tests to show that treatment is needed.”

By the time we become adults, most of use have been infected by CMV. However in most cases our immune systems hold it in check. In stem cell transplant patients, however, the immune system is replaced with those of a donor after receiving sizable doses of chemotherapy. During this period, long-dormant viruses, such as CMV, can reactivate and cause CMV disease. CMV is a type of herpes virus. Herpes viruses do a very good job of keeping a low profile and hiding in various types of cells. Only by treating with an effective anti-viral drug can CMV disease be thwarted.

In this Phase 2 clinical trial, 230 stem cell transplant recipients at 27 different centers across the United States were randomly assigned to either the oral CMX001 group to the placebo group. All patients took the drugs or placebos after their bone marrow transplant procedure and the drugs or placebos were taken for 9-11 weeks.

Those patients that took 100 milligrams of CMX001 twice a week, 10% had a CMV event in which CMV was detectable in the blood and the symptoms of CMV disease appeared. However, 37% of those patients who took the placebo had a CMV event. The most common side effect was diarrhea, which is no surprise given the fragile state of these patients.

“The results show the effectiveness of CMX001 in preventing CMV infections in this group of patients,” said Marty. “Because CMX001 is known to be active against other herpes viruses and against adenoviruses that sometimes affect transplant patients, it may be useful as a preventative or treatmentagent for those infections as well.”

See New England Journal of Medicine, 2013; 369(13): 1227.

Increasing Engraftment Rates of Umbilical Cord Blood Transplantations


Harvard Stem Cell Institute (HSCI) researchers have published initial results of a Phase Ib human clinical trial of a therapeutic that has the potential to improve the success of blood stem cell transplantation. This publication marks a success for the HSCI and their ability to carry a discovery from the lab bench to the clinic. This was actually the mandate for the HSCI when it was founded.

This Phase 1b safety study was published in the journal Blood, and it included 12 adult patients who underwent umbilical cord blood transplantation for leukemia or lymphoma at the Dana Farber Cancer Institute and Massachusetts General Hospital. Each patient received two umbilical cord blood units; one of which was untreated and another that was treated with a small molecule called 16,16 dimethyl prostaglandin E2 (dmPGE2). The immune systems of all 12 patients were successfully reconstituted and their bone marrow tissues were able to make blood cells. However, 10 of the 12 patients had blood formation that was solely derived from those umbilical cord blood cells that had been treated with dmPGE2.

This clinical test is now entering Phase II, during which the HSCI scientists will determine the efficacy of this treatment in 60 patients at 8 different medical centers. They expect results from this trial within 18-24 months.

The success of the HSCI depended on collaborations with scientists at different Harvard-affiliated institutions. These collaborations included 1) Leonard Zon, chair of the HSCI Executive Committee and Professor of Stem Cell and Regenerative Biology at Harvard, and his colleagues, 2) Dana-Farber Cancer Institute and Massachusetts General Hospital, led by hematologic oncologist and HSCI Affiliated Faculty member Corey Cutler, and 3) Fate Therapeutics, Inc., a San Diego-based biopharmaceutical company of which Zon is a founder, sponsored the Investigational New Drug application, under which the clinical program was conducted, and translated the research findings from the laboratory into the clinical setting.

“The exciting part of this was the laboratory, industry, and clinical collaboration, because one would not expect that much close interplay in a very exploratory trial,” Cutler said. “The fact that we were able to translate someone’s scientific discovery from down the hall into a patient just a few hundred yards away is the beauty of working here.”

Gastroenterologists have been interested in dmPGE2 for decades, because it has the ability to protect the intestinal lining from stress. However, its ability to amplify stem cell populations was identified in 2005 during a chemical screen exposing 5,000 known drugs to zebrafish embryos. Wolfram Goessling, MD, PhD, and Trista North, PhD former Zon postdoctoral fellows, were involved in that work.

“We were interested in finding a chemical that could amplify blood stem cells and we realized looking at zebrafish embryos that you could actually see blood stem cells budding from the animal’s aorta,” Zon said. “So, we elected to add chemicals to the water of fish embryos, and when we took them out and stained the aortas for blood stem cells, there was one of the chemicals, which is this 16,16 dimethyl prostaglandin E2, that gave an incredible expansion of stem cells—about a 300 to 400 percent increase.”

The dramatic effects of this molecule on blood stem cells causes Zon, who practices as a pediatric hematologist, consider how this prostaglandin could be applied to bone marrow transplantation. Bone marrow transplantations are often used to treat blood cancers, including leukemia and lymphoma. Bone marrow contains the body’s most plentiful reservoir of blood stem cells, and so patients with these conditions may be given bone marrow transplants to reconstitute their immune systems after their cancer-ravaged bone marrow has been wiped out with chemotherapy and radiation.

Zon designed a preclinical experiment, similar to the one later done with cord blood patients, in which mice undergoing bone marrow transplants received two sets of competing bone marrow stem cells, one set treated with dmPGE2 and a second untreated set.

“What we found was the bone marrow stem cells that were treated with prostaglandin, even for just two hours, had a four times better chance of engrafting in the recipient’s marrow after transplant,” he said. “I was very excited to move this into the clinic because I knew it was an interesting molecule.”

Zon and his team’s then visited the Dana Farber Cancer Institute (DFCI). There, they presented the mouse research at bone marrow transplant rounds and found physicians interested in giving the prostaglandin to patients.

“We basically sat down in a room and we brainstormed a clinical trial based on their scientific discovery, right then and there,” said Farber oncologist Corey Cutler. “They knew that it was something they could bring to the clinic, but they just didn’t know where it would fit. We said, if this molecule does what you say it does, significant utility would lie in umbilical cord blood transplants.”

A cord blood transplant is similar to a bone marrow transplant, but the blood stem cells are not from an adult donor but from the umbilical cord blood of a newborn. The degree of tissue matching is less in an umbilical cord blood transplant than in a bone marrow transplant. The umbilical cord stem cells are young and incipient and the immune system simply does not recognize them as readily as adult cells. Therefore, potentially fatal graft-versus-host disease is less common with umbilical cord blood transplants. About 10-20 percent of stem cell transplantation procedures now use umbilical cord blood. However the main disadvantage of umbilical cord blood transplantations is that the cord blood contains uses smaller amounts of cells, which makes engraftment is more difficult.

Umbilical cord blood transplants fail about 10 percent of the time. Therefore, increasing the procedure’s success would significantly help patients who do not have adult bone marrow donors, including a disproportionate number of non-Caucasian patients in North America. Increasing the engraftment rate would also allow the use of smaller umbilical cord blood units that are potentially better matches to their recipients, increasing the number of donations that go on to help patients.

Fate Therapeutics received the first green light from the US Food and Drug Administration, and the DFCI Institutional Review Board for this clinical trial. Umbilical cord blood processing was done by Dana-Farber’s Cell Manipulation Core Facility, directed by HSCI Executive Committee member Jerome Ritz, MD. There was a stumbling block in that once the human trial was underway with the first nine patients in that the protocol in use, which was developed in mice, did not translate to improved engraftment in humans.

“The initial results were very disappointing,” Cutler said. “We went back to the drawing board and tried to figure out why, and it turned out some of the laboratory-based conditions were simply not optimized, and that was largely because when you do something in the lab, the conditions are a little bit different than when you do it in a human.”

Fate Therapeutics discovered that the human cord blood was being handled at temperatures that were too cold (4-degrees Celsius) for the prostaglandin to biologically activate the stem cells. Therefore even after prostaglandin treatment, the umbilical cord blood did not show enhanced engraftment rates. Fate further demonstrated that performing the incubation of the hematopoietic stem cells at 37-degrees Celsius and increasing the incubation time from 1 hour to 2 hours elicited a much stronger gene and protein expression response that correlated with improved engraftment in animal models.

In running a second cohort of the Phase Ib trial, which included 12 patients, dmPGE2 appeared to enhance the engraftment properties of the blood stem cells in humans and was deemed safe to continue into Phase II. “It’s probably the most exciting thing I’ve ever done,” Zon said. “Basically, to watch something come from your laboratory and then go all the way to a clinical trial is quite remarkable and very satisfying.”